Illegal trade of wildlife is growing internationally and is worth more than USD$20 billion per year. DNA technologies are well suited to detect and provide evidence for cases of illicit wildlife trade yet many of the methods have not been verified for forensic applications and the diverse range of methods employed can be confusing for forensic practitioners. In this review, we describe the various genetic techniques used to provide evidence for wildlife cases and thereby exhibit the diversity of forensic questions that can be addressed using currently available genetic technologies. We emphasise that the genetic technologies to provide evidence for wildlife cases are already available, but that the research underpinning their use in forensics is lacking. Finally we advocate and encourage greater collaboration of forensic scientists with conservation geneticists to develop research programs for phylogenetic, phylogeography and population genetics studies to jointly benefit conservation and management of traded species and to provide a scientific basis for the development of forensic methods for the regulation and policing of wildlife trade.

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.© 2011 Blackwell Publishing Ltd.

Historical mitochondrial introgression causes differences between a species' mitochondrial gene genealogy and its nuclear gene genealogy, making tree-based species delineation ambiguous. Using sequence data from one mitochondrial gene (cytochrome b) and three nuclear genes (introns), we examined the evolutionary history of four high elevation Tibetan megophryid frog species, Scutiger boulengeri, Scutiger glandulatus, Scutiger mammatus and Scutiger tuberculatus. The three nuclear genes shared a similar history but the mitochondrial gene tree suggested a drastically different evolutionary scenario. The conflicts between them were explained by multiple episodes of mitochondrial introgression events via historical interspecific hybridization. 'Foreign' mitochondrial genomes might have been fixed in populations and extended through a large portion of the species' distribution. Some hybridization events were probably as old as 10 Myr, while others were recent. An F(1) hybrid was also identified. Historical hybridization events among the four species appeared to be persistent and were not restricted to the period of Pleistocene glaciation, as in several other well-studied cases. Furthermore, hybridization involved several species and occurred in multiple directions, and there was no indication of one mitochondrial genome being superior to others. In addition, incomplete lineage sorting resulting from budding speciation may have also explained some discrepancies between the mitochondrial DNA and nuclear gene trees. Combining all evidences, the former 'Scutiger mammatus' appeared to be two species, including a new species. With the availability of a wide range of highly variable nuclear gene markers, we recommend using a combination of mitochondrial gene and multiple nuclear genes to reveal a complete species history.

Terrain use is a fundamental factor of wildlife habitat and is closely related to other environmental factors. To assess the impact of cattle and horse on wildlife, we conducted field surveys in Wanglang National Nature Reserve and analyzed terrain preferences. We also deployed infrared cameras and historical biodiversity data to evaluate the risk of disturbance to key protected species based on altitudinal distribution of livestock. Results showed: (1) Both horse and cattle preferred low altitude habitats with low slopes, close to water with half to full sun aspects. However, there was also significant differences in terrain use between these two domestic animals. (2) The areas with the most frequent disturbance by livestock in the Wanglang Nature Reserve were Youyizhigou and Zhenggou in Zhugencha, and Yangdonggou in Dawodang. (3) Based on historical data, takin (Budorcas taxicolor tibetana) is likely the most influenced wildlife species, as the degree of overlap with livestock along altitudinal gradients is very high. Infrared camera data showed that areas frequented by livestock had fewer takin individuals recorded, indicating a negative relationship due to co-existence. Based on our findings, we suggest, (1) nature reserve should focus livestock in two core areas of Zhugencha (Youyizhigou, Zhenggou and Baishagou) and Dawodang (Yangdonggou and Waicepo) by monitoring the population and distribution of livestock in these areas; (2) they should prohibit the dispersal and distribution of livestock towards higher elevations; (3) the frequency with which local herdsman feed livestock salt needs to be controlled; (4) the strength of law enforcement towards illegal herds should be elevated.

地形是栖息地的基本要素, 从地形评价动物的空间利用特征能够掌握动物的分布规律并进行预测。为掌握保护区内牲畜的空间利用特征, 并评价它们对主要保护动物的潜在影响, 我们于2018年5-11月调查了王朗国家级自然保护区内牛和马的分布, 并结合红外相机监测结果及历史监测数据进行了分析和评价。结果表明: (1)虽然两种牲畜均偏好低海拔、低坡度、光照良好(半阳坡、阳坡)、距水源近的栖息地, 但它们在地形利用上存在显著差异; (2)牲畜活动最频繁的三条沟分别是竹根岔右一支沟、竹根岔正沟和大窝凼洋洞沟, 且呈现不同的干扰特征; (3)基于监测数据, 羚牛(Budorcas taxicolor tibetana)可能是保护区内最易受牛马活动威胁的保护动物。红外相机监测结果显示, 羚牛沿海拔分布现状可能是回避牲畜密集区域的结果。基于本研究, 我们建议: (1)保护区重点关注竹根岔(右一支沟、正沟、白沙沟)、大窝凼(洋洞沟、外侧坡)两个核心区的牲畜活动情况, 并尽快针对放牧采取措施。例如, 持续监测重点干扰区域牲畜的种群数量和空间分布趋势。(2)严格限制牲畜继续向高海拔栖息地入侵。(3)管控放牧投盐等干扰的发生频率。(4)加强执法力度, 防止牲畜对保护区带来的干扰持续和扩大, 威胁物种安全。

Biodiversity analyses can greatly benefit from coherent species delimitation schemes and up-to-date distribution data. In this article, we have made the daring attempt to delimit and map described and undescribed lineages of anuran amphibians in the Eastern Palaearctic (EP) region in its broad sense. Through a literature review, we have evaluated the species status considering reproductive isolation and genetic divergence, combined with an extensive occurrence dataset (nearly 85k localities). Altogether 274 native species from 46 genera and ten families were retrieved, plus eight additional species introduced from other realms. Independent hotspots of species richness were concentrated in southern Tibet (Medog County), the circum-Sichuan Basin region, Taiwan, the Korean Peninsula and the main Japanese islands. Phylogeographic breaks responsible for recent in situ speciation events were shared around the Sichuan Mountains, across Honshu and between the Ryukyu Island groups, but not across shallow water bodies like the Yellow Sea and the Taiwan Strait. Anuran compositions suggested to restrict the zoogeographical limits of the EP to East Asia. In a rapidly evolving field, our study provides a checkpoint to appreciate patterns of species diversity in the EP under a single, spatially explicit, species delimitation framework that integrates phylogeographic data in taxonomic research.

We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

The Himalaya presents an outstanding geologically active orogen and biodiversity hotspot. However, our understanding of the historical biogeography of its fauna is far from comprehensive. Many taxa are commonly assumed to have originated from China-Indochina and dispersed westward along the Himalayan chain. Alternatively, the "Tibetan-origin hypothesis" suggests primary diversification of lineages in Paleo-Tibet, and secondary diversification along the slopes of the later uplifted Greater Himalaya. We test these hypotheses in high-mountain megophryid anurans (Scutiger). Extensive sampling from High Asia, and analyses of mitochondrial (2839 bp) and nuclear DNA (2208 bp), using Bayesian and Maximum likelihood phylogenetics, suggest that the Himalayan species form a distinct clade, possibly older than those from the eastern Himalaya-Tibet orogen. While immigration from China-Indochina cannot be excluded, our data may indicate that Himalayan Scutiger originated to the north of the Himalaya by colonization from Paleo-Tibet and then date back to the Oligocene. High intraspecific diversity of Scutiger implies limited migration across mountains and drainages along the Himalaya. While our study strengthens support for a "Tibetan-origin hypothesis", current sampling (10/22 species; 1 revalidated: S. occidentalis) remains insufficient to draw final conclusions on Scutiger but urges comparative phylogeographers to test alternative, geologically supported hypotheses for a true future understanding of Himalayan biogeography.

The toad genus Oreolalax is widely distributed in southwest China and northern Vietnam. A new species of the genus is described from Sichuan Province, China. Phylogenetic analyses based on the mitochondrial 12S rRNA and 16S rRNA gene sequences supported the new species as an independent clade clustered into the clade also containing O. nanjiangensis and O. chuanbeiensis. The new species can be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 51.2–64.2 mm in males); head broad; tympanum hidden; interorbital region with dark triangular pattern; belly with marbling; male lacking spines on lip margin; spiny patches on chest small with thick sparse spines in male; nuptial spines thick and sparse; tibio-tarsal articulation reaching beyond nostril when leg stretched forward; toe webbing at base.

大熊猫是我国的特产动物,古时被列为贡品,在近代被视为国宝。新中国成立以前,由于乱捕滥猎等因素,该物种已处于濒危状态。解放以后,我国政府于20 世纪50 年代开始了自然保护区建设,并于20 世纪60 年建立了卧龙等5 个大熊猫自然保护区,并将大熊猫列为禁猎动物。20 世纪70 年代我国进行了第一次全国大熊猫资源调查,基于调查结果新建了佛坪、唐家河、蜂桶寨等自然保护区,使有关大熊猫的自然保护区总数达到了13 个。1985 -1988 年全国第二次大熊猫资源调查结果显示,野生大熊猫种群数量较第一次减少了54 ﹪。进入90 年代,全国大熊猫保护区增至36 个,迄今共建立了63 个与保护大熊猫这一珍稀物种相关的自然保护区。全国大熊猫栖息地已由80 年代衰落期时22 220 km2 增加至34 000 km2,其中分布在保护区内的栖息地面积达29 000 km2,有效地保护了约85% 的大熊猫栖息地总面积以及约50% 的野生大熊猫种群数量。现存野生大熊猫种群数量估计约2 000只,因密度稀疏,发展空间大。目前,国家主管部门已出台了各种就地与迁地保护措施,只需各方通力合作,野生大熊猫的未来将是光明的。

Developing effective policies for biodiversity conservation and restoration policies requires spatially and temporally explicit data on distribution of species and habitats. Remote sensing provides an effective technical tool to meet this requirement. In recent years, the rapid development of integrated multi-platform, multi-scale, multi-mode remote sensing technology the implementation of integrated remote sensing observations across space-air-field provides novel opportunities for biodiversity monitoring. In this paper, we review the main methods of remote sensing that aids biodiversity monitoring and assess existing remote sensing observation experiments. We found that current methods of biodiversity monitoring using remotely-sensed data lacked the support of space-air-field integrated observations and the existing space-air-field integrated observations did not include biodiversity parameters. The Wanglang integrated observation and experiment station for mountain ecological remote sensing illustrates the potential to integrate experimental station-based and space-air-field integrated observations for biodiversity monitoring. Our review highlights that integrating direct observations with remote sensing can provide spatio-temporally explicit information on species and habitats and improve the informed monitoring of biodiversity.

科学制定生物多样性保护和恢复政策, 需要空间上连续、时间上高频的物种和生境分布以及物种迁移信息支持, 遥感是目前能满足该要求的有效技术手段。近年来, 遥感平台和载荷技术高速发展, 综合多平台、多尺度、多模式遥感技术, 开展基于站点的星空地一体化遥感观测试验, 可以对地表进行时空多维度、立体连续观测, 为生物多样性遥感监测提供了新的契机。本文总结了使用遥感技术监测生物多样性的主要方法, 回顾了典型的星空地一体化遥感观测试验。综述以往研究发现, 一方面, 现有遥感试验还缺少对生物多样性直接监测指标的观测, 另一方面, 生物多样性遥感监测方法也缺少星空地多维立体观测平台的支撑, 亟需加强两者的融合, 开展基于站点的生物多样性星空地一体化遥感监测研究。以设于我国四川王朗大熊猫国家级自然保护区内的王朗山地生态遥感综合观测试验站为例, 展示了星空地一体化遥感综合观测试验平台在生物多样性监测中的应用潜力。星空地一体化遥感观测可以提供物种和生境的综合定量信息, 与生态模型有机结合, 可以刻画生物多样性的时空格局与动态过程, 有助于挖掘过程机理, 提高生物多样性监测的信息化水平。

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

To investigate the resources, diversity and distribution of culturable mucoromycotan fungi in the Greater Khingan Mountains, 279 samples including dry branches and fallen leaves, humus, soil and faeces were collected from nine representative counties and cities. A total of 1,153 strains of mucoromycotan fungi were isolated using a dilution plate, cutting and direct incubation methods. Among the 1,153 strains, 706 representatives were analyzed on the basis of fungal molecular barcode ITS rDNA. These mucoromycotan fungi were classified into 3 orders, 8 families, 10 genera and 38 species. Dominant genera were Mortierella, Umbelopsis, and Mucor, while dominant species were Mortierella amoeboidea, Mucor hiemalis, and Umbelopsis isabellina. This paper also summarized all 26 genera of Mucoromycotina and Mortierellomycotina reported in China, and the distribution of the dominant genera and species of the Greater Khinggan Mountains were analyzed. In addition, regional distribution of all genera was analyzed with regard to three major ecological regions (Eastern wet and semi-humid ecological regions, northwestern arid and semi-arid ecological regions and the Tibetan Plateau alpine ecological regions). The results showed that nine genera were distributed in all the three regions. For endemic genera, nine were found in the Eastern wet and semi-humid ecological region, only one in northwestern arid and semi-arid ecological region, and none in the Tibetan Plateau alpine ecological regions.

为查明大兴安岭地区可培养毛霉门真菌的资源、多样性及其分布, 本研究选取9个代表市县, 采集了279份枯枝落叶、腐殖质、土壤和粪便样品, 采用稀释平板挑取法、稀释平板切块法和样品直接培养挑取法进行分离培养。通过形态初步观察鉴定, 共得到毛霉门真菌1,153株。选取代表性的菌株706株, 基于真菌分子条形码ITS rDNA进行分子系统发育多样性分析, 明确了毛霉门真菌总计3目8科10属38种。优势属为被孢霉属(Mortierella)、伞形霉属(Umbelopsis)和毛霉属(Mucor), 优势种为类变形被孢霉(Mortierella amoeboidea)、冻土毛霉(Mucor hiemalis)和深黄伞形霉(Umbelopsis isabellina)。本文同时汇总了全国已报道毛霉亚门和被孢霉亚门共计26属的分布, 分析了大兴安岭优势属和优势种在全国的分布。按三大主要生态区(东部湿润、半湿润生态区, 西北干旱、半干旱生态区和青藏高原高寒生态区)对所有属进行区域分析, 结果表明: 有9个属在3个大区都有分布; 对特有属而言, 东部湿润、半湿润生态区发现9个, 西北干旱、半干旱生态区仅有1个, 青藏高原高寒生态区未发现。

The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed).We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.

Species identification was carried out by nucleotide sequence analysis of the cytochrome b (cytb) gene. The aim of the study was to identify biological specimens from diverse vertebrate animals by extracting and amplifying DNA from 44 different animal species covering the 5 major vertebrate groups (i.e. mammals, birds, reptiles, amphibians and fishes). The sequences derived were used to identify the biological origin of the samples by aligning to cytb gene sequence entries in nucleotide databases using the program BLAST. All sequences were submitted to the GenBank including new species which were not observed in the databases. The applicability of this method to the forensic field is demonstrated by simulated casework conditions where different types of samples including problematic specimens such as hair, bone samples, bristles and feathers were investigated to identify the species.

The extant amphibians are one of the most diverse radiations of terrestrial vertebrates (>6800 species). Despite much recent focus on their conservation, diversification, and systematics, no previous phylogeny for the group has contained more than 522 species. However, numerous studies with limited taxon sampling have generated large amounts of partially overlapping sequence data for many species. Here, we combine these data and produce a novel estimate of extant amphibian phylogeny, containing 2871 species (∼40% of the known extant species) from 432 genera (∼85% of the ∼500 currently recognized extant genera). Each sampled species contains up to 12,712 bp from 12 genes (three mitochondrial, nine nuclear), with an average of 2563 bp per species. This data set provides strong support for many groups recognized in previous studies, but it also suggests non-monophyly for several currently recognized families, particularly in hyloid frogs (e.g., Ceratophryidae, Cycloramphidae, Leptodactylidae, Strabomantidae). To correct these and other problems, we provide a revised classification of extant amphibians for taxa traditionally delimited at the family and subfamily levels. This new taxonomy includes several families not recognized in current classifications (e.g., Alsodidae, Batrachylidae, Rhinodermatidae, Odontophrynidae, Telmatobiidae), but which are strongly supported and important for avoiding non-monophyly of current families. Finally, this study provides further evidence that the supermatrix approach provides an effective strategy for inferring large-scale phylogenies using the combined results of previous studies, despite many taxa having extensive missing data.Copyright © 2011 Elsevier Inc. All rights reserved.

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese-breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour-joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan-specific cryptic species from trans-Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans-Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.© 2014 John Wiley & Sons Ltd.

Advances in fecal DNA technology have expanded its usages in molecular ecology, especially in genetic assessment of wild animals. The technology allows researchers to understand ecological issues without contacting, disturbing, or even seeing animals, thus avoided invasions to the animal studied, and greatly promotes studies in molecular ecology of wild animals. Although this technology could yield poor DNA and relatively high genotyping errors in its early stages, these problems have been overcome gradually with the technological achievements being made in the past 25 years. Nowadays, fecal DNA technology yields good DNA and low genotyping errors, allowing researchers to address questions in reliability. Here, we share our knowledge about technological pitfalls on fecal sampling, preservation, DNA extraction, PCR, and genotyping in detail, and discuss opportunities and challenges of its applications, aiming to increase the power and role of the technology in molecular ecology of wild animals.<br>

Large carnivores play an important role in the regulation of food-web structure and ecosystem functioning. However, large carnivores face serious threats that have caused declines in their populations and geographic ranges due to habitat loss and degradation, hunting, human disturbance and pathogen transmission. Conservation of large carnivore species richness and population size has become a pressing issue and an important research focus of conservation biology. The western Sichuan Plateau, located at the intersection of the mountains of southwest China and the eastern margin of the Tibetan Plateau, is a global biodiversity hotspot and has high carnivore species richness. However, increasing human activities may exacerbate the destruction of local flora and fauna, thereby threatening the survival of wild carnivores. Information on species composition and dietary habits can improve our understanding of the structure and function of the ecosystem and food-web relationships in the study area. In addition, species composition and dietary habits are of great significance for understanding multi-species coexistence mechanisms and preserving biodiversity. This study collected carnivore fecal samples from Xinlong and Shiqu counties in the Ganzi Tibetan Autonomous Prefecture, Sichuan Province. DNA was then extracted from the samples and the species was identified based on DNA sequences and DNA barcoding techniques. Seven carnivores were identified, including five large carnivores (Canis lupus, Ursus arctos, Panthera pardus, P. uncia and Canis lupus familiaris) and two medium and small-sized carnivores (Prionailurus bengalensis and Vulpes vulpes). Using fecal DNA, high-throughput sequencing and metabarcoding, we conducted diet analysis for the seven carnivores and found 28 different food molecular operational taxonomic units (MOTUs), including 19 mammals, eight birds and one fish species. The predominant prey categories of wolves, dogs and brown bears were ungulates. The domestic yak (Bos grunniens) was the most frequently identified prey species. Small mammals such as rodents and lagomorphs accounted for a significant proportion in the diets of leopard cats and red foxes, The most frequent prey of this category of carnivore were the Chinese scrub vole (Neodon irene) and plateau pika (Ochotona curzoniae). In addition, leopards and snow leopards mainly fed on the Chinese goral (Naemorhedus griseus) and blue sheep (Pseudois nayaur), respectively. Our study highlights the utility of fecal DNA and metabarcoding technique in fast carnivore surveys and high-throughput diet analysis, and provides a technical reference and guidance for future biodiversity surveys and food-web studies.

在陆地生态系统中, 大型食肉动物对于稳定食物网结构和生态系统功能有重要作用。在世界范围内, 由于栖息地丧失和破碎化、猎杀、人类活动干扰以及病原体的传播, 大型食肉动物生存正面临严重威胁, 多种食肉动物地理分布范围及种群数量大幅度缩减。如何有效保护大型食肉动物物种多样性及种群已经成为世界关注的焦点问题和保护生物学的重要研究方向。川西高原地处我国西南山地与青藏高原东缘交界地带, 属于世界生物多样性热点地区, 是世界大型食肉动物物种最丰富的地区之一, 而日益增强的人类活动可能会加剧对当地动植物资源的破坏, 进而威胁野生食肉动物的生存。获得准确的物种多样性信息及食肉动物食性数据有助于深入了解该地区生态系统结构及食物网关系, 对研究物种共存机制及生物多样性保护有重要意义。本研究通过从四川甘孜藏族自治州新龙县和石渠县野外采集的食肉动物粪便样品中提取DNA, 利用DNA条形码进行物种鉴定, 快速获得该地区食肉动物物种构成信息。38份粪便样品经鉴定来自于7种食肉动物, 分别为5种大型食肉动物(狼Canis lupus、棕熊Ursus arctos、豹Panthera pardus、雪豹P. unica、狗Canis lupus familiaris)和2种中小型食肉动物(豹猫Prionailurus bengalensis、赤狐Vulpes vulpes)。进一步利用高通量测序和宏条形码技术对7种食肉动物粪便中的食物DNA进行精准食性分析, 得到包含19种哺乳类、8种鸟类和1种鱼类共计28个不同的食物分子可操作分类单元(molecular operational taxonomic unit, MOTU)。结果显示, 狼、狗、棕熊最主要的食物来源为偶蹄目动物, 其中取食频率最高的物种为家牦牛(Bos grunniens); 而豹猫和赤狐食物中小型哺乳动物如啮齿目和兔形目占重要比例, 其中高原松田鼠(Neodon irene)和高原鼠兔(Ochotona curzoniae)被取食频率最高。豹和雪豹的食物分别为偶蹄目的中华斑羚(Naemorhedus griseus)和岩羊(Pseudois nayaur)。本研究显示了粪便DNA及宏条形码技术在食肉动物多样性快速调查及高通量精确食性分析中的应用前景, 并为此类研究提供了技术路线的有力借鉴。

The relative efficiencies of the maximum parsimony (MP) and distance-matrix methods in obtaining the correct tree (topology) were studied by using computer simulation. The distance-matrix methods examined are the neighbor-joining, distance-Wagner, Tateno et al. modified Farris, Faith, and Li methods. In the computer simulation, six or eight DNA sequences were assumed to evolve following a given model tree, and the evolutionary changes of the sequences were followed. Both constant and varying rates of nucleotide substitution were considered. From the sequences thus obtained, phylogenetic trees were constructed using the six tree-making methods and compared with the model (true) tree. This process was repeated 300 times for each different set of parameters. The results obtained indicate that when the number of nucleotide substitutions per site is small and a relatively small number of nucleotides are used, the probability of obtaining the correct topology (P1) is generally lower in the MP method than in the distance-matrix methods. The P1 value for the MP method increases with increasing number of nucleotides but is still generally lower than the value for the NJ or DW method. Essentially the same conclusion was obtained whether or not the rate of nucleotide substitution was constant or whether or not a transition bias in nucleotide substitution existed. The relatively poor performance of the MP method for these cases is due to the fact that information from singular sites is not used in this method. The MP method also showed a relatively low P1 value when the model of varying rate of nucleotide substitution was used and the number of substitutions per site was large. However, the MP method often produced cases in which the correct tree was one of several equally parsimonious trees. When these cases were included in the class of "success," the MP method performed better than the other methods, provided that the number of nucleotide substitutions per site was small.

The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain a large collection of methods and tools of computational molecular evolution. Here, we describe new additions that make MEGA a more comprehensive tool for building timetrees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods for estimating divergence times and confidence intervals are implemented to use probability densities for calibration constraints for node-dating and sequence sampling dates for tip-dating analyses, which will be supported by new options for tagging sequences with spatiotemporal sampling information, an expanded interactive Node Calibrations Editor, and an extended Tree Explorer to display timetrees. We have now added a Bayesian method for estimating neutral evolutionary probabilities of alleles in a species using multispecies sequence alignments and a machine learning method to test for the autocorrelation of evolutionary rates in phylogenies. The computer memory requirements for the maximum likelihood analysis are reduced significantly through reprogramming, and the graphical user interface (GUI) has been made more responsive and interactive for very big datasets. These enhancements will improve the user experience, quality of results, and the pace of biological discovery. Natively compiled GUI and command-line versions of MEGA11 are available for Microsoft Windows, Linux, and macOS from www.megasoftware.net.© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

We used infrared camera to monitor wild animals in Wanglang National Nature Reserve. Our goal was to estimate species diversity, the relationship between the number of cameras and number of species, the relationship between the camera days and number of species, and the relative abundance of species. Across 42 locations, we recorded a total of 1,793 images in which we found 25 species of wildlife. Species found in our camera traps included three national first-class protected species such as giant panda (Ailuropoda melanoleuca), Sichuan takin (Budorcas tibetanus) and buff-throated partridge (Tetraophasis szechenyii). We also recorded eight national second-class protected wild animals such as the Asiatic black bear (Ursus thibetanus), yellow-throated marten (Martes flavigula), Chinese serow (Capricornis milneedwardsii), Chinese goral (Naemorhedus griseus). These 25 species were captured when we increased the number of trap-days increased to 23 or any single the trap was placed for at least 180 days. Furthermore the blood pheasant (Ithaginis cruentus) (29.28) and the tufted deer (Elaphodus cephalophus) (15.78) had the highest relative abundances in the reserve. The relative abundance index for the giant panda was 8.09 and the indices for temminck’s tragopan (Tragopan temminckii), Chinese serow and buff-throated partridge was between 2 and 5. The Chinese goral, koklass pheasant (Pucrasia macrolopha), Asiatic black bear, Sichuan takin and blue eared-pheasant (Crossoptilon auritum) had the lowest relative abundance values (all < 1). In summary, we found camera-trapping to be an effective method for wildlife survey. Our findings suggest that less abundant species need more camera trapping effort. Nature reserves are important for protecting habitats of endangered species.

本研究以四川王朗国家级自然保护区为研究区域, 利用红外相机对保护区内的主要野生动物进行了初步调查, 分析了该区域的物种多样性现状、相机数量和相机工作日与物种数量间的关系以及物种的相对丰富度。结果表明: 42台红外相机共拍摄到物种独立照片1,793张, 鉴定出野生动物25种, 包括大熊猫(Ailuropoda melanoleuca)、四川羚牛(Budorcas tibetanus)和黄喉雉鹑(Tetraophasis szechenyii) 3种国家一级重点保护野生动物, 黑熊(Ursus thibetanus)、黄喉貂(Martes flavigula)、中华鬣羚(Capricornis milneedwardsii)、中华斑羚(Naemorhedus griseus)等8种国家二级重点保护野生动物。在相机数量增加到23台的时候拍摄到了本次记录的全部25种野生动物, 并且在单台相机工作日达到180天时, 物种数达到饱和。保护区内物种相对丰富度最高的是血雉(Ithaginis cruentus)(29.28)和毛冠鹿(Elaphodus cephalophus)(15.78); 大熊猫的相对丰富度为8.09; 红腹角雉(Tragopan temminckii)、中华鬣羚和黄喉雉鹑的相对丰富度在2-5之间; 中华斑羚、勺鸡(Pucrasia macrolopha)、黑熊、四川羚牛和蓝马鸡(Crossoptilon auritum)的相对丰富度最低, 不到1。综上所述, 红外相机能够有效地对野生动物资源进行监测调查, 对于相对丰富度较低的物种需要投入更多的精力, 这些物种的栖息地保护对于自然保护区的发展至关重要。

<p id="p00015"><strong>Aims</strong> Biodiversity monitoring is the foundation of conservation work in national parks. Systematic conservation planning and effective management actions within these parks highly rely on an in-depth evaluation of biodiversity metrics. In China, the Giant Panda National Park (GPNP) is one of the first five national parks. To establish baseline metrics of mammal and bird diversity in GPNP, we conducted a meta-analysis based on published camera-trapping data. <br><strong> Methods</strong> We systematically searched academic publications, project reports, and news articles that reported on wildlife camera-trapping studies between 2005 and 2020 in GPNP. We also conducted a questionnaire survey on the history and results of camera-trap monitoring projects within the protected areas of the region. These data were compiled for statistical analysis. <br><strong> Result</strong> Between 2005 and 2020, 71 wild mammal species belonging to 6 orders, 22 families and 55 genera, and 232 wild bird species belonging to 13 orders, 45 families and 132 genera were recorded via camera-trap monitoring in 51 protected areas within GPNP. Among the four mountain ranges within GPNP (i.e., Mts. Qinling, Mts. Minshan, Mts. Qionglai and Mts. Xiangling), the species richness of large- and medium-sized terrestrial mammals and birds was the highest in Mts. Qionglai and Mts. Minshan (40 mammal and 12 pheasant species for each) and the lowest in Mts. Xiangling (25 mammal and 7 pheasant species). The recorded number of target species in individual protected areas was positively correlated with the area of protected area, sampling effort (measured as number of camera-days), and the camera station elevation range. The numbers of species recorded in national protected areas (28 &#x000b1; 8.3, mean &#x000b1; SD) were significantly higher than those in provincial protected areas (19 &#x000b1; 8.9). Four large carnivores of Felidae and Canidae (leopard <i>Panthera pardus</i>, snow leopard <i>P. uncia</i>, wolf <i>Canis lupus</i> and dhole <i>Cuon alpinus</i>) were recorded in GPNP, primarily from Mts. Qinling and Mts. Qionglai, while no large carnivores were recorded within the park in Mts. Minshan and only one wolf was recorded in Mts. Xiangling. <br><strong> Conclusion</strong> The previous protected area network and camera-trapping monitoring network prior to the establishment of GPNP have already accumulated a high quantity of data on wild mammals and birds in this region. These data provide a reliable baseline biodiversity inventory for the pilot and construction phases of GPNP. In light of these results, GPNP should design and implement a standardized wildlife monitoring system to further provide additional data for future evaluations of park management, decision-making, and conservation effectiveness.</p>

生物多样性监测是国家公园保护的核心基础。大熊猫国家公园是我国首批5个国家公园之一, 系统的保护规划与有效的管理行动均有赖于对区内生物多样性本底、现状与动态的深入了解。为了解大熊猫国家公园范围内兽类与鸟类多样性本底与现状, 本研究系统检索了该区域内2005-2020年基于红外相机调查技术的野生动物研究论文、项目报告以及新闻报道, 并对区内原有保护地的红外相机监测历史与结果进行了问卷调查。结果表明, 2005-2020年期间, 在大熊猫国家公园范围内51个保护地的红外相机调查与监测中, 共记录到分属6目22科55属的71种野生兽类与分属13目45科132属的232种野生鸟类。在国家公园所覆盖的秦岭、岷山、邛崃山、相岭4大山系中, 邛崃和岷山记录到的大中型地栖鸟兽物种多样性最高(均为兽类40种, 鸟类12种), 相岭最低(兽类25种, 鸟类7种)。单个保护地中记录到的大中型地栖鸟兽物种数量与保护地面积、红外相机有效工作日及相机位点的海拔跨度均呈正相关, 国家级保护地中记录到的物种数(28 &#x000b1; 8.3, mean &#x000b1; SD)显著高于省级保护地(19 &#x000b1; 8.9)。在大熊猫国家公园内共记录到猫科与犬科的4种大型食肉动物, 即豹(Panthera pardus)、雪豹(P. uncia)、狼(Canis lupus)和豺(Cuon alpinus), 主要来自于秦岭山系和邛崃山系, 而国家公园内的岷山山系则没有记录到大型食肉动物, 相岭山系中仅有1次狼的记录。本研究结果显示, 大熊猫国家公园内前期已经建立起的自然保护地网络与红外相机监测体系已积累大量区内野生兽类与鸟类的基础数据, 为国家公园的试点与建设提供了生物多样性编目与监测方面的可靠本底。在这些前期工作的基础上, 大熊猫国家公园应进一步规划、建设标准化的野生动物监测体系, 为今后国家公园的管理决策、成效评估提供坚实的科学支撑。

Background & Aim: Analyzing biodiversity status requires multi-spatial scale, continuous monitoring across different ecosystems due to its heterogenous nature in both space and time. Therefore, monitoring networks are necessary for biodiversity conservation research. Biodiversity monitoring networks at the global, regional, and national scales, represented by GEO BON and APBON, have flourished. China has established a long-term monitoring network for ecosystems and species at the national scale. and the China Biodiversity Observation and Research Network (Sino BON) was launched in 2013 with strong support from the Chinese Academy of Sciences and the Ministry of Finance. Review Results: Sino BON includes 10 subnetworks specialized at monitoring animals, plants and microbes and an additional network for near-ground remote sensing, which covers 30 main sites and 60 affiliated sites in China. Currently, Sino BON has created a research platform for multi-trophic interactions among soil microorganisms, insects, large mammals, underground forests to forest canopies. This platform provides an understanding of biodiversity change and its driving factors at the national level and may be used in protecting biodiversity and sustainable utilization of biological resources. Perspectives: For further progresses, monitoring technology, monitoring areas, data standards and integrated information platforms require further development.

生物多样性强烈的时空尺度依赖性和多层次性决定了生物多样性现状与变量的分析需要在不同生态系统进行多空间尺度、全面和连续的监测。因此, 构建生物多样性研究监测网络是生物多样性保护和研究的基础工作。近年来, 对地观测组织-生物多样性观测网络(GEO BON)、亚太生物多样性监测网络(APBON)等全球、区域以及国家尺度的生物多样性监测网络蓬勃发展。中国陆续在国家尺度上建立了针对生态系统和物种的长期监测网络, 其中, 中国生物多样性监测与研究网络(China Biodiversity Observation and Research Network, Sino BON)于2013年启动建设, 在我国主要生态系统和环境梯度设置30个监测主点和60个监测辅点, 目前已建成10个专项网对动物、植物和微生物进行监测, 并建立了以数据标准与汇交、近地面遥感为核心的综合监测中心。Sino BON打造了从地下、地面到森林林冠的多尺度、多类群(功能群)以及多营养级交互为重点的监测与研究平台, 为理解生物多样性变化趋势及其驱动因素、研究生物多样性维持机制, 以及国家履行《生物多样性公约》、保护生物多样性和生物资源提供详实可靠的生物多样性变化数据。为进一步支撑国家生物多样性治理能力、深化全球多样性保护合作, 我国生物多样性监测亟需在监测技术、监测区域、数据标准、综合信息平台等方向谋求更大的发展。

Understanding the diets of carnivores is essential for resolving food web interactions and the population dynamics of both prey and predators and for designing effective strategies for species and ecosystem conservation. As effective predators, wild felids play important roles in various ecosystems, but relatively little is known about the dietary habits of many species, primarily owing to their elusive behavior. We used a DNA-based method to analyze the vertebrates and plants constituting the diet of leopard cats (Prionailurus bengalensis) in the temperate forests of the mountains of southwest China, a global biodiversity hotspot. DNA extracted from leopard cat scats was amplified with primers targeting either the vertebrate mitochondrial 12S rRNA gene (N = 25 scats) or the psbCL region of the chloroplast genome of vascular plants (N = 42 scats). The polymerase chain reaction products were sequenced and prey taxa were assigned based on sequence similarity. We identified a total of 16 taxa of vertebrate prey, with pikas (in 76% of the scats) and rodents (40%) predominating. Plant material belonging to 12 taxa was found in 76% of the samples, and the genus Solanum and subfamily Rosoideae were the most frequently detected plant taxa. The frequency of occurrence of identified plant taxa differed between the spring–summer and fall–winter months. Thus, the leopard cats in our study area have a diversified diet and are highly flexible in adapting their foraging behavior to the locally available prey. Our data suggest that preserving their natural prey base dominated by pikas and rodents may be vital for the subsistence of local populations. The high species resolution and detection sensitivity of the DNA-based method we used in this study make it a powerful and efficient tool for fine-scale analysis of complex diets.

The loss of biodiversity resulting from extinctions is receiving increasing attention. Over several thousands of animal species have been evaluated and recognized as endangered species. Inbreeding depression has been demonstrated in many wild animal species. Here we sequenced 655-978 bp mitochodrial D-loop region of 32 individuals from four regional giant panda populations. Sixteen haplotypes were observed. AMOVE analysis demonstrated that genetic differentiation was not significant in the overall population, except the Qingling population. The current panda population may recover from a recent severe bottleneck that occurred about 43 000 years ago. Combining with our results on two endangered snub-nosed monkey species and one common hare species, different scenarios for low genetic variation have been discussed. Our results suggest that low genetic variation does not necessary result from a recent bottleneck, and it is not necessarily an indication of the level of endangerment.